CN105062933A - Lactobacillus reuteri and application thereof - Google Patents
Lactobacillus reuteri and application thereof Download PDFInfo
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- CN105062933A CN105062933A CN201510579432.7A CN201510579432A CN105062933A CN 105062933 A CN105062933 A CN 105062933A CN 201510579432 A CN201510579432 A CN 201510579432A CN 105062933 A CN105062933 A CN 105062933A
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Abstract
The invention relates to lactobacillus reuteri and application thereof. Lactobacillus reuteri is lactobacillus reuteri LZ262 which is preserved in the Common Microorganism Center of the China Committee for Culture Collection of Microorganisms on 13 August, 2015 with the preservation number of CGMCCNo.11240. Lactobacillus reuteri LZ262 is from the intestinal tract of a healthy pig, can be colonized in a human body and an animal body, and has higher tolerance to gastric acid and bile salt; in addition, lactobacillus reuteri LZ262 has a better suppression effect on Escherichia coli, and has a higher survival rate after spray-drying, and higher fermentation density is achieved by improving a fermentation formula; the strain has a better effect of synergistic growth with other types of lactic acid bacteria, can be used for food and animal feed additives, and can also be widely applied to other food and biological products.
Description
Technical field
The present invention relates to field of biological, particularly relate to a kind of lactobacillus reuteri and application thereof.
Background technology
Lactobacillus reuteri, also lactobacillus reuteri is claimed, with the mankind, the life of animal is in close relations, that one is common in people, pig, milk-acid bacteria in poultry and other animal intestinals, by stomach and be colonizated in enteron aisle play beneficial effect, such as have the balance maintaining intestinal flora, promote absorption of nutrient ingredients, alleviate the several functions such as the formation of lactose intolerance and inhibition tumor cell, current research reports that more Application Areas comprises food, feed, health care, makeup, beauty treatment, aquaculture, industrial and agricultural production, biological preservative, probiotics preparation etc.
The different physiological roles of lactobacillus reuteri, can be used as the source of host's nitrogen.But when thalline is acidproof, does not enter enteron aisle during bile tolerance scarce capacity, its effect will be lost.
Therefore, these prebiotic effects be played, first must can tolerate the digestive process of human intestines and stomach, gastric juice and bile can be resisted to its killing action.It is 90min that Berrada etc. report food emptying under one's belt time, and in stomach, pH value is 3.0, minimumly reaches 1.3, and human small intestine's bile fluid concentration is between 0.03%--0.3%.
Physiological condition seed selection one strain from health pig enteron aisle that the present invention is directed to human body and animal stomach can tolerate the lactobacillus reuteri of human body and animal hydrochloric acid in gastric juice and gallbladder salinity, thus can field planting in human body and animal body, plays prebiotic effect.This bacterial strain has good restraining effect to colibacillus simultaneously, can play the function of regulating intestinal canal microorganism species.
According to current relevant report, obtain the method for lactobacillus reuteri of resistance to human body and animal cholate having three kinds: one to be that two is screen from traditional fermented food, and three is tame existing bacterial classification from occurring in nature screening.The physiological condition that the present invention is directed to human body and animal stomach screens, and cultivates the lactobacillus reuteri that a strain can tolerate human body and animal body hydrochloric acid in gastric juice and gallbladder salinity, thus likely field planting in human body and animal body, play its prebiotic effect.
Summary of the invention
The object of the present invention is to provide a kind of lactobacillus reuteri deriving from health pig enteron aisle, make this lactobacillus reuteri to acid, cholate, have good tolerance, have stronger inhibition to intestinal bacteria, thus be more suitable for practicality.
Another object of the present invention is to the application providing a kind of lactobacillus reuteri, this lactobacillus reuteri is widely used.
The object of the invention to solve the technical problems is achieved by the following technical solution.According to a kind of lactobacillus reuteri that the present invention proposes, also claim lactobacillus reuteri, it is lactobacillus reuteri LZ262, and preserving number is CGMCCNo.11240, and preservation date is on 08 14th, 2015.Depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center.
The object of the invention to solve the technical problems also can be applied to the following technical measures to achieve further.
A first aspect of the present invention, described lactobacillus reuteri fermention medium has higher viable bacteria amount.
A second aspect of the present invention, described lactobacillus reuteri has good tolerance to acid, cholate, can field planting in human body and animal body.
A third aspect of the present invention, described lactobacillus reuteri has higher survival after spraying dry, has certain temperature tolerance, can participate in granulation directly in Pelleting.
A fourth aspect of the present invention, described lactobacillus reuteri has stronger restraining effect to intestinal bacteria.
A fifth aspect of the present invention, described lactobacillus reuteri has good compatibility to appointment milk-acid bacteria.Described one of lactobacillus reuteri LZ262 and following milk-acid bacteria or more than one are composite, have symplastic growth effect; Lactobacterium acidophilum (Lactobacillusacidophius); Lactobacillus delbrueckii kind (Lactobacillusdelbrueckiisubsp.bulgaricus); Casei subspecies (Lactobacilluscaseisubsp.Casei); Thermophilus streptococcus (Streptococcusthermophius); Streptococcus acidi lactici (Streptococcuslactis); Lactococcus lactis subsp.lactis (Lactococcuslactissubsp.lactis); Bifidobacterium animalis subspecies (Bifidobacteriumanimalissubsp.lactis); Plant lactobacillus (Lactobacillusplantarum).
A sixth aspect of the present invention, described lactobacillus reuteri has stronger product proteolytic enzyme ability.
The object of the invention to solve the technical problems also realizes by the following technical solutions.The application of a kind of lactobacillus reuteri proposed according to the present invention, wherein said lactobacillus reuteri LZ262 is used for animal feedstuff additive, antibacterial liquid, food, healthcare products and aquaculture.
The application of aforesaid lactobacillus reuteri, wherein said lactobacillus reuteri LZ262 is that the ratio between wherein said lactobacillus reuteri LZ262 and other probiotic bacterium is: lactobacillus reuteri LZ262: Lactobacillus acidophilus: lactobacterium casei: plant lactobacillus=(1 ~ 2): (3 ~ 5): (2 ~ 3): (1 ~ 2) by described lactobacillus reuteri LZ262 and other probiotic bacterium assembly when being used for animal feedstuff additive.
The application of aforesaid lactobacillus reuteri, wherein said lactobacillus reuteri LZ262 carries out growing and fattening pigs for animal feedstuff additive and feeds, result be effectively promote growing and fattening pigs growth, reduce the diarrhea rate of piglet, suppress pernicious bacteria and stimulate intestine immunity reaction aspect to play a significant role, reduce the mortality ratio of pig.
By technique scheme, the present invention at least has following advantages: the present invention is separated and obtains, through being accredited as lactobacillus reuteri from healthy raw material; The lactobacillus reuteri obtained more than invented has good tolerance to human body and animal body gastrointestinal environment, and proteolytic enzyme can be produced help digest, there is stronger temperature tolerance, good symplastic growth effect is had with milk-acid bacteria, expand the range of application of this bacterium, fermentation has higher viable bacteria amount, decreases fermentation costs.
In addition, described lactobacillus reuteri has good restraining effect to intestinal bacteria, has higher fermentation density, have higher survival rate after spraying dry by improvement fermentating formula.
Accompanying drawing explanation
Fig. 1 is the opticmicroscope figure of lactobacillus reuteri LZ262 of the present invention.
Embodiment
Example is: this lactobacillus reuteri, it is lactobacillus reuteri (Lactobacillusreuteri) LZ262, preserving number is CGMCCNo.11240, and preservation date is on 08 14th, 2015, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center.
The Isolation and Identification method of lactobacillus reuteri LZ262 is as follows:
1, separation method
To take in sample (health pig enteron aisle) 5g to 45ml sterile buffered saline (adding appropriate granulated glass sphere) as mother liquor, concussion 10min fully mixes, gradient dilution is done with sterile buffered saline, select the gradient that three to five suitable, draw 0.1ml in MRS substratum, carry out coating separation, each dilution gradient does two repetitions.Culture dish is put into 37 DEG C of incubators cultivations and is selected smooth, convex garden, neat in edge, white or oyster white bacterium colony microscopy after 48 ~ 72 hours.This separation, purifying are carried out in MRS substratum again, until bacterium colony Economical Purification by above-mentioned steps to doubtful bacterium colony.
Morphological Identification: gramstaining, cellular form, with or without gemma
Physiology and biochemistry is identified: catalase, oxydase, and carbohydrate produces acid
Molecular biology identification: 16SrRNA sequential analysis
2. experimental result
2.1 physio-biochemical characteristics and morphological specificity
The physio-biochemical characteristics of table 1LZ262
2.216SrRNA sequencing results
ATACATGCAGTCGTACGCACTGGCCCAACTGATTGATGGTGCTTGCACCTGATTGACGATGGATCACCAGTGAGTGGCGGACGGGTGAGTAACACGTAGGTAACCTGCCCCGGAGCGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAACAACAAAAGCCACATGGCTTTTGTTTGAAAGATGGCTTTGGCTATCACTCTGGGATGGACCTGCGGTGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACAATGGAACTGAGACACGGTCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTGGAGAAGAACGTGCGTGAGAGTAACTGTTCACGCAGTGACGGTATCCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTGCTTAGGTCTGATGTGAAAGCCTTCGGCTTAACCGAAGAAGTGCATCGGAAACCGGGCGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTGCGCTAACCTTAGAGATAAGGCGTTCCCTTCGGGGACGCAATGACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCAAGCTCGCGAGAGTAAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTTGTAACGCCCAAAGTCGGT
According to above-mentioned 16SrRNA sequencing results (sequence table), identify that LZ262 is lactobacillus reuteri.
3. qualification result
In conjunction with above qualification result, LZ262 of the present invention is lactobacillus reuteri.
The optimization of lactobacillus reuteri LZ262 substratum and cultural method thereof
1 materials and methods
1.1 test strain
Lactobacillus reuteri LZ262, is located away from health pig enteron aisle.
1.2 materials and equipment
Tryptones: Beijing extensive and profound in meaning star biotechnology limited liability company biochemical reagents
Soy peptone: Beijing extensive and profound in meaning star biotechnology limited liability company biochemical reagents
Yeast extract paste: Beijing extensive and profound in meaning star biotechnology limited liability company biochemical reagents
Extractum carnis: Beijing extensive and profound in meaning star biotechnology limited liability company biochemical reagents
Pork liver leaching powder: Beijing extensive and profound in meaning star biotechnology limited liability company biochemical reagents
Angel peptone FP101: Angel Yeast Co., Ltd's biochemical reagents
Angel Yeast leaching powder FM902: Angel Yeast Co., Ltd's biochemical reagents
Beef peptone: Beijing bispin microbiological culture media products factory biochemical reagents
Casein peptone: Beijing extensive and profound in meaning star biotechnology limited liability company biochemical reagents
K
2hPO
4: Beijing Chemical Plant's analytical pure
KH
2pO
4: Beijing Chemical Plant's analytical pure
Lactose: Beijing extensive and profound in meaning star biotechnology limited liability company analytical pure
Semi-lactosi: Chemical Reagent Co., Ltd., Sinopharm Group's biochemical reagents
Starch: Tianjin great Mao chemical reagent factory analytical pure
Sucrose: Beijing Chemical Plant's analytical pure
Maltose: Beijing extensive and profound in meaning star biotechnology limited liability company biochemical reagents
Glucose: good fortune analytical pure in morning
Manganous sulfate: Xi Long chemical plant, Shantou, Guangdong city analytical pure
Magnesium sulfate: Chemical Reagent Co., Ltd., Sinopharm Group's analytical pure
Sodium acetate: Xilong Chemical Co., Ltd
Tween 80: Tianjin good fortune chemical reagent factory in morning
Citric acid diamines: Xilong Chemical Co., Ltd's analytical pure
Triammonium citrate: Xilong Chemical Co., Ltd's analytical pure
752 type spectrophotometers: Shanghai Spectrum Apparatus Co., Ltd.
Vertical pressure steam sterilizer: Jiangyin Binjiang Medical Equipment Co., Ltd. LSB50L-I
Bechtop: 100 grades, Wujiang Lan Lin air-purification unit company limited
Analytical balance: Sai Duolisi scientific instrument company limited BSA124S-Cw
Microorganism fermentation tank: Shanghai wide generation biotechnology equipment company limited GS-8000-10L
Table-type high-speed refrigerated centrifuge: Xiang Zhi whizzer Instrument Ltd. model 090613
Small spraying drying instrument: SY_6000
1.3 substratum and culture condition
Activation and counting are cultivated and are adopted MRS substratum, peptone 10g, extractum carnis 10g, yeast extract paste 5g, KH
2pO
42g, citric acid diamines 2g, sodium acetate 2g, glucose 20g, Tween801mL, MgSO
47H
2o0.58g, MnSO
44H2O0.25g, is settled to 1L with distilled water, adjusts pH6.2 ~ 6.4,121 DEG C of sterilizing 15min.
Based on MRS substratum, add different somatomedin according to test design, inoculum size 2%-5% accesses in 150mL liquid nutrient medium, and 37 DEG C of quiescent culture 24h, survey its growth curve.
1.4 method
1.4.1 the selection in different carbon source, nitrogenous source, phosphorus source
On the basis of MRS liquid nutrient medium, add 2% (w/v) lactose, sucrose, glucose, fructose, semi-lactosi, starch, maltose respectively as different carbon source; Add respectively 2.5% (w/v) Tryptones, soy peptone, yeast extract paste, extractum carnis, Angel peptone FP101, Angel Yeast leaching powder FM902, pork liver leaching powder, beef liver leaching powder, beef peptone as different nitrogen sources, other components are all identical.Lactobacillus reuteri in the medium after access activation, cultivates 24h, after diluting 10 times, measures bacterium liquid OD value under 600nm wavelength under the condition of 37 DEG C, and with MRS substratum for contrast, more different carbon sources, nitrogenous source are on the impact of growth of lactobacillus reuteri.
1.4.2 the optimization of substratum
Adopt orthogonal experimental design optimize carbon source, nitrogenous source consumption due to, level of factor is as shown in table 1.Wherein A factor is maltose, and B factor is Angel Yeast powder FM902, and C factor is Angel peptone PF101, and D factor is that Australia wins star pork liver leaching powder, measures bacterium liquid OD value.
Table 1 orthogonal test level of factor
1.4.3OD pH-value determination pH
With 722 type spectrophotometers, after bacterium liquid is diluted 10 times, do reference with the substratum not connecing bacterium under the same terms, under 600nm wavelength, measure bacterium liquid absorbancy.
1.4.4 enumeration
With MRS solid medium for counting substratum, take suitable extension rate, be poured on flat board, count after 37 DEG C of cultivation 48h.
2 results and analysis
2.1 carbon sources are on the impact of growth of lactobacillus reuteri
Through contrast four kinds of different carbon source lactose, sucrose, glucose, maltose, semi-lactosi, starch on the impact of growth of lactobacillus reuteri, find that the effect of maltose is comparatively remarkable.
2.2 nitrogenous sources are on the impact of growth of lactobacillus reuteri
With MRS substratum for contrast, contrast four kinds of single nitrogenous source Tryptoness, soy peptone, yeast extract pastes, extractum carnis, beef peptone, pork liver leaching powder, beef liver leaching powder, casein peptone, to the growth effect of lactobacillus reuteri.As can be seen from experimental result, the growth of lactobacillus reuteri when being single nitrogenous source with pork liver leaching powder, OD value a little more than contrast, apparently higher than the growth of lactobacillus reuteri in other single nitrogenous source; Lactobacillus reuteri growth result when taking Tryptones as nitrogenous source is poor, is unfavorable for the growth of lactobacillus reuteri, well can not promotes growth when adding separately.
The optimization of 2.3 lactobacillus reuteri substratum
According to the result of single factor experiment, design orthogonal test, factor A, B, C, D are respectively maltose, Angel Yeast powder, Angel peptone, pork liver leaching powder, and with light absorption value (OD600nm) for evaluation index, measurement result and corresponding range analysis are in table 2.
Table 2 orthogonal test level of factor
As can be seen from Table 2, influence factor size is arranged as A>D>C>B, i.e. maltose > pork liver leaching powder > Angel peptone > Angel Yeast powder; During maltose influence factor level most high-content 2.5%, light absorption value (OD600nm) is the highest, the content of pork liver leaching powder is The more the better, consider fix on 2% comparatively suitable, Angel peptone content is between 0.7-0.8%, Angel Yeast powder influence factor is minimum, fixes between 0.3-0.5%.
3 inspections: detect viable bacteria amount
| Numbering | Viable bacteria amount cfu/ml |
| MRS | 9.2*10 8 |
| A1B3C2D3 | 7.6*10 9 |
4 brief summaries
Lactobacillus reuteri LZ262 substratum after employing optimization of orthogonal test, viable bacteria amount verifies as 7.6*10
9cfu/ml, higher than the 9.2*10 of MRS
8cfu/ml.
5 lactobacillus reuteri LZ262 cultural methods
5.1 test strain
Lactobacillus reuteri LZ262
5.2 materials and equipment
The same
5.3 substratum and culture condition
Activation and counting are cultivated and are adopted MRS substratum: peptone 10g, extractum carnis 10g, yeast extract paste 5g, KH
2pO
42g, citric acid diamines 2g, sodium acetate 2g, glucose 20g, Tween801mL, MgSO
47H
2o0.58g, MnSO
44H
2o0.25g, is settled to 1L with distilled water, adjusts pH6.2 ~ 6.4,121 DEG C of sterilizing 15min.
Fermention medium: pork liver leaching powder 20g, Angel peptone PF1017g, Angel Yeast leaching powder PM9023g, K
2hPO
42g, Trisodium Citrate 2g, sodium acetate 5g, maltose 25g, Tween801mL, MgSO
47H
2o0.58g, MnSO
44H
2o0.25g, is settled to 1L with distilled water, adjusts pH6.5,115 DEG C of sterilizing 20min.
Frozen-dried protective agent prescription: skimming milk 10%-20%, lactose 0.5%-1.5%, sucrose 5%-10%, Sodium Glutamate 0.1%-1%.
Spraying protective material formula: skimming milk 5%-15%, sucrose 3%-10%, lactose 1%-2%, trehalose 3%-10%.
5.4 method
5.4.1 cultural method
5.4.1.1 glycerine seed activation gets glycerol stocks strain inoculation in the liquid test tube of MRS, (35 DEG C-40 DEG C) quiescent culture 10-15 hour.
5.4.1.2 seed culture presses the inoculum size of 3%-6%, and get appropriate amount from activation bacterium liquid, access is equipped with in the 250ml triangular flask of 150ml4# liquid nutrient medium, (35 DEG C-40 DEG C) quiescent culture.
5.4.1.3 fermentation culture presses the inoculum size of 3%-6%, and access is equipped with in the 10L fermentor tank of substratum, and adjustment temperature 35 DEG C-40 DEG C, rotating speed 50-100r/min, initial p H6.0-7.0 cultivate.
5.4.1.4 stop fermentation when after bacteria growing 15-24h, fermentor tank is cooled to about 20 DEG C, lower tank.
5.4.1.5 lyophilize 4 degrees Celsius of 4000-8000 leave heart 5-10min, and remove supernatant, buffering salt washes bacterium, and 4 degrees Celsius of 4000-8000 leave the heart, remove supernatant, collect bacterium mud, with protective material by 1:(3-6) be mixed and carry out lyophilize.
5.4.1.6 freeze-drying method: bacterium mud is mixed with protective material, by a certain amount of loading cold-trap pre-freeze to subzero 30-40 DEG C, propose cold-trap setting lyophilize parameter and carry out drying, parameter is as follows:
| Temperature parameter (DEG C) | Time parameter (hour) |
| -25--35 | 1-4 |
| -15--25 | 1-4 |
| -5-0 | 4-6 |
| 1-5 | 1-8 |
| 15-25 | 1-4 |
| 25-35 | 1-4 |
5.4.1.7 spraying dry: 4 degrees Celsius of 4000-8000 leave heart 5-10min, and remove supernatant, buffering salt washes bacterium, 4 degrees Celsius of 4000-8000 leave the heart, remove supernatant, collect bacterium mud, with protective material by 1:(4-9) be mixed and carry out spraying dry.
5.4.1.8 spray drying process: bacterium mud mixes with protective material, the preheating of instrument washing and sterilizing, inlet temperature setting 150-170 DEG C, air outlet temperature is 65-75 DEG C, peristaltic pump rotating speed 10-20, collects chilled product.
5.4.2 measuring method
5.4.2.1 live bacterial count: adopt dilution pour plate counting process to measure viable count.
5.4.2.2 survival rate measures: Bacterium lacticum survival rate (%)=(viable count before the viable count/spraying dry after spraying dry) × 100%
5.4.2.3 determination of moisture: adopt furnace drying method to measure product moisture content, the baking oven that the drying basin that 2-3g sample is housed puts into 105 DEG C is heated, until weight, repeat aforesaid operations, until the difference of twice consecutive weighings quality is no more than 0.0005g (GB/T5413.8-1997).
M1: the initial mass adding drying basin after sample, unit is g;
M2: sample dries the rear minimum mass obtained of weighing for twice, and unit is g;
M3: sample quality, unit is g.
5.4.2.4 survival rate measures:
Survival rate (%)=(viable count/spraying after spraying or freeze-drying or viable count before freeze-drying) × 100%
5.4.2.5 high temperature resistant mensuration: get a certain amount of atomized powder and be placed in baking oven 85 DEG C of xeothermic 10min, measure its viable count.
6. result
| Project | Freeze-drying | Spraying |
| Viable count | 7.85×10 11 | 6.05×10 10 |
| Moisture | 2.8％ | 4.1％ |
| Survival rate | 95％ | 58％ |
| High temperature resistant mensuration | - | 27.4％ |
Lactobacillus reuteri acid and bile salt tolerance is tested
1. experimental technique
1.1 cholate resistance test
The modified MRS nutrient solution 37 DEG C of lactobacillus reuteri LZ262 sterilizing cultivates 15h.By activated strains nutrient solution with 2% inoculum size access containing in the aseptic MRS liquid culture medium of various biliary salt concn (gallbladder salinity for 0.2%, 0.3%, 0.5%, 1.0%, 1.6%), simultaneously with not containing the modified MRS nutrient solution of cholate for contrast.Sampling and measuring viable count after cultivating 0h, 2h, 4h, 6h, 24h at 37 DEG C.
2.2 sour tolerance tests
Lactobacillus reuteri LZ262 aseptic modified MRS nutrient solution 37 DEG C cultivates 15h, by activation nutrient solution with 2% inoculum size access different PH aseptic modified MRS in (PH is respectively 1.3,2.0,3.0), simultaneously with the nutrient solution of PH6.5 for contrast.37 DEG C of cultivations, respectively at 0min, 30min, 60min, 90min, 120min. sampling and measuring viable bacteria.
3. experimental result
3.1 bile tolerance experimental results
Lactobacillus reuteri LZ262 is to the tolerance of various biliary salt concn in table 3, and to its unrestraint effect when gallbladder salinity is less than or equal to 0.3%, in 6h, viable count constantly increases, and in the gallbladder salinity of 0.5%-1.6%, growth has certain tolerance.
Table 3 lactobacillus reuteri LZ262 is to the tolerance of different concns cholate
| 0h | 2h | 4h | 6h | 24h | |
| CK | 6.6×10 7 | 2.7×10 8 | 3.38×10 8 | 1.21×10 9 | 6.20×10 9 |
| 0.2％ | 6.14×10 7 | 1.58×10 8 | 1.37×10 8 | 5.87×10 8 | 5.76×10 9 |
| 0.3％ | 5.95×10 7 | 8.30×10 7 | 1.30×10 8 | 6.19×10 8 | 1.03×10 9 |
| 0.5％ | 4.88×10 7 | 8.25×10 7 | 2.70×10 8 | 2.73×10 8 | 5.25×10 8 |
| 1％ | 4.89×10 7 | 4.48×10 7 | 2.77×10 7 | 2.28×10 7 | 1.85×10 6 |
| 1.6％ | 3.48×10 7 | 2.46×10 7 | 1.40×10 7 | 2.74×10 6 | 1.05×10 6 |
3.2 acidproof experimental results
Lactobacillus reuteri LZ262 viable count change in different acid nutrient solutions, in table 4, under PH2.0 condition, is cultivated and within 2 hours, is still had a large amount of viable bacteria to exist, illustrate that this bacterial strain has very strong tolerance to acid.
Table 4 lactobacillus reuteri LZ262 is to the tolerance of acid
| 0min | 30min | 60min | 90min | 120min | |
| PH6.5 | 5.13×10 7 | 5.18×10 7 | 5.35×10 7 | 6.80×10 7 | 1.02×10 8 |
| PH 3.0 | 4.85×10 7 | 4.80×10 7 | 4.23×10 7 | 4.38×10 7 | 3.28×10 7 |
| PH 2.0 | 5.05×10 7 | 4.20×10 7 | 3.50×10 7 | 1.50×10 7 | 7.82×10 6 |
| PH 1.3 | 4.52×10 7 | 4.05×10 7 | 2.25×10 7 | 6.00×10 6 | 1.00×10 6 |
Brief summary
This experiment is separated the lactobacillus reuteri LZ262 obtained has good tolerance to acid, cholate and room temperature.In the stomach of people, pH value is between 2-3, and after eating acidic food, pH value is minimum reaches 1.5.Lactobacillus reuteri LZ262 can be survived 2 hours in the sour environment of PH=2 and PH=1.3, only have dropped an order of magnitude; And under the acidic conditions of PH=3, after 2 hours, the order of magnitude of viable count does not change.And food (especially fluid) is relatively short by the time of stomach, after general 1 ~ 2h, just enter small intestine.So the acid resistance of lactobacillus reuteri LZ262 can ensure that it has a considerable amount of viable bacteria to arrive enteron aisle by stomach.
The gallbladder salinity of human small intestine is between 0.03-0.3%, and do not have restraining effect to lactobacillus reuteri LZ262 when gallbladder salinity is less than 0.3% in this experiment, in 6h, viable count constantly increases.Lactobacillus reuteri LZ262 has very strong tolerance to cholate, can arrive large intestine by small intestine.
Lactobacillus reuteri LZ262 is to the biological antagonist of intestinal bacteria, milk-acid bacteria
1. materials and methods
1.1 bacterial classification
Lactobacillus reuteri LZ262, intestinal bacteria, Lactobacterium acidophilum, lactobacterium casei, streptococcus acidi lactici, Lactobacillus delbrueckii kind, bifidobacterium breve, bifidobacterium adolescentis, bifidumbacterium bifidum all comes from this laboratory.
1.2 substratum
MRS liquid and solid medium, tomato juice solid and liquid nutrient medium, nutrient broth solid and liquid nutrient medium
1.3 actication of culture
Lactobacillus reuteri activates: the lactobacillus reuteri being placed in low temperature refrigerator is put into 40 ~ 50 DEG C of water-baths and thaws, and aseptically, draws 0.2ml and is inoculated in MRS liquid nutrient medium, 37 DEG C of constant temperature culture 12 ~ 14h, 2 ~ 3 generations of activation.
The activation of milk-acid bacteria: same to lactobacillus reuteri, lactobacillus adopts the activation of MRS liquid nutrient medium, and streptococcus acidi lactici adopts the activation of tomato juice substratum.
Colibacillary activation: by the intestinal bacteria be placed in refrigerator according to 2% inoculum size be transferred to liquid nutrient broth substratum, 37 DEG C of constant temperature culture 12h, activated for two generations.
1.4 coating nutrient agar medium plates
The intestinal bacteria activated coating method is applied on nutrient agar plate uniformly, requires that viable bacteria amount during coating is 10
6cfu/ml.
1.5 tilt-pour processes cultivate milk-acid bacteria
The lactobacillus activated and streptococcus acidi lactici are layered on MRS flat board with tilt-pour process respectively uniformly and on tomato juice solid plate, require that viable bacteria amount during coating is 10
6cfu/ml.
The preparation of 1.6 lactobacillus reuteri fermented liquids
The lactobacillus reuteri fermented liquid activated is adopted the method for low-temperature centrifugation, prepare fermented supernatant fluid, centrifugal condition is: 4 DEG C, 5000-8000rpm, 5-10min.
1.7 lactobacillus reuteri fermented liquid Bacteriostatic Effects
Odontothrips loti: by the nutrient agar plate of the indicator of completing, MRS solid plate and tomato juice solid plate seasoning 30min, aseptically Oxford cup (internal diameter is 6mm) is evenly positioned on flat board again, press down a little, make itself and plate culture medium contact surface tight, then 0.2ml fermented supernatant fluid is drawn in cup, 37 DEG C of constant temperature culture 24h, observe the size of inhibition zone, and measure antibacterial circle diameter.Each sample do three parallel, results averaged.Control group adds MRS liquid nutrient medium 0.2ml in the cup of Oxford, and culture condition is identical.
1.8 experimental result
Result shows, most of Bacterium lacticum has restraining effect to intestinal bacteria, and lactobacillus reuteri fungistatic effect of the present invention is particularly evident, and concrete outcome is in table 5.
Experiment shows, the lactobacillus reuteri belonging to the present invention and most of milk-acid bacteria can symplastic growths, and this expands the range of application of lactobacillus reuteri, and concrete outcome is in table 6.
Table 5 milk-acid bacteria and colibacillary antagonistic effect
| Strain name | Antibacterial circle diameter (mm) |
| LZ262 lactobacillus reuteri | 23 |
| YDB14 bifidobacterium breve | 16 |
| YDB109 bifidobacterium adolescentis | 16 |
| YDB114 bifidobacterium adolescentis | 13 |
| YDB120 bifidumbacterium bifidum | 14 |
| YDL197 lactobacterium casei | 10 |
| YDL200 Lactobacterium acidophilum | 13 |
| YDL124 Lactobacterium acidophilum | 7 |
| YDL165 Lactobacterium acidophilum | 16 |
| BJYL132 plant lactobacillus Bacterium lacticum | 25 |
| YDL192 Lactobacterium acidophilum | 15 |
The compatibility (antagonistic effect) of table 6 lactobacillus reuteri and milk-acid bacteria
| Strain name | Test-results | Strain name | Test-results |
| YDL200 Lactobacterium acidophilum | － | YDL18 lactobacterium casei | － |
| YDL124 Lactobacterium acidophilum | － | YDL126 lactobacterium casei | － |
| YDL163 Lactobacterium acidophilum | － | YDS151 thermophilus streptococcus | － |
| YDL192 Lactobacterium acidophilum | － | BJYL132 plant lactobacillus | － |
| YDL137 Lactobacterium acidophilum | － | YDS194 thermophilus streptococcus | － |
| YDL184 lactobacillus bulgaricus De Shi breast subspecies | － | YDS011 Lactococcus lactis subsp.lactis | － |
| YDL162 lactobacillus bulgaricus De Shi breast subspecies | － | YDS125 streptococcus acidi lactici | － |
| YDL197 lactobacterium casei | － | B174 bifidobacterium animalis subspecies | - |
| YDL108 lactobacterium casei | － | YDS170 thermophilus streptococcus | － |
Lactobacillus reuteri LZ262 is as follows for the preparation of the method for fodder additives:
Lactobacillus reuteri LZ262 prebiotic effect of the present invention is obvious, can with other probiotic bacterium assembly, in animal-feed.Test shows, composite probiotics preparations effectively can attack poison from colibacillary, improves colibacillary resistibility.Ratio between each probiotic bacterium is: lactobacillus reuteri LZ262: Lactobacillus acidophilus: lactobacterium casei: plant lactobacillus=(1 ~ 2): (3 ~ 5): (2 ~ 3): (1 ~ 2).
The product proteolytic enzyme ability that lactobacillus reuteri LZ262 is stronger
1, experiment material and instrument
Tyrosine, sodium carbonate, Folin reagent, casein, trichoroacetic acid(TCA), water-bath, spectrophotometer, whizzer.
2, experimental technique
(1) formulation of tyrosine typical curve
The tyrosine standardized solution (0 of preparation different concns, 20,40,60,80,100 μ gml-1), get the tyrosine standardized solution 1.00mL of different concns respectively, respectively add the sodium carbonate solution 5.00mL of 0.4molml-1, forint diluent 1.00mL, be placed in 40 DEG C of water-baths and develop the color 20min, take out, measure its absorbancy OD660 (not contain the pipe of tyrosine for blank) respectively with spectrophotometer.
The tyrosine standardized solution of different concns and the variation relation of OD660
| Tyrosine concentration (μ gml-1) | 10 | 20 | 30 | 40 | 50 | 60 | 80 |
| OD660 | 0.101 | 0.21 | 0.308 | 0.409 | 0.513 | 0.625 | 0.81 |
With OD value for X-coordinate, with the concentration of tyrosine for ordinate zou drawing standard curve, obtain typical curve equation: y=98.22x-0.3288 (R2=0.9994)
(2) fermented liquid proteinase activity is surveyed
1. bacterial strain in Bechtop, be seeded in the 4# substratum of 50ML respectively by the inoculum size of 2%-3%, cultivate in 37 DEG C of incubators.
2. the centrifugal 20min of fermented liquid 3500r/min, gets supernatant liquor and is crude enzyme liquid.
3. first 2% casein is put into 40 DEG C of waters bath with thermostatic control, preheating 2min.Get 1.0ml crude enzyme liquid preheating 2min, add the casein 1.0ml after preheating, shake up, and be accurately incubated 10min at 40 DEG C.
4. add 0.4mol/L trichoroacetic acid(TCA) 2.0ml again, shake up rear continuation water-bath and place 20min to precipitate residual protein, the centrifugal 10min of 3500r/min, gets supernatant liquor 1.0ml,
5. at 40 DEG C, 0.4mol/L sodium carbonate solution 5.0ml and 1.0ml forint phenol reagent is added, insulation 20min.
6. colorimetric under 660nm wavelength, surveys its absorbancy.(when measuring blank, adding casein contrary with the order of trichoroacetic acid(TCA)).
Note: proteinase activity method of calculation:
Enzyme activity (U/ml)=(OD sample determination-OD blank test) × K × 4/10 × N
In formula, K=98.22; 4:4 milliliter reaction solution is got 1mL and is measured (namely 4 times); N: the multiple of enzyme liquid dilution, this experiment is 5 times; 10: reaction 10min.
The definition of enzyme activity unit: produce 1 μ g tyrosine at 40 DEG C of per minute caseinhydrolysates, be defined as 1 proteinase activity.
Experimental result
Lactobacillus reuteri cultivates 18 hours, and protease activity reaches 8.4602U/ml.
Lactobacillus reuteri LZ262 is to the administering transgenic of sucking piglets
1. experiment material
Selection large white sow parity and birth piglet 12 nest that the product date is close in advance, be divided into control group and experimental group at random.
2. test place: pig farm, Beijing
3. experimental technique
Control group: nurse according to a conventional method and feed to wean.
Experimental group: piglet of feeding in 0-7 day after piglet birth to be fed agent based on the liquid of Roy's formula Bacterium lacticum LZ262, and every day drinks 4ml to every piglet, serve on three days; 7 age in days-28 ages in days wean, the lactobacillus reuteri LZ262 powdery adding 0.2% in sucking pig material is fed agent.
4. detect
4.1 weigh: weigh piglet birth individual weight and wean individual weight.
4.2 feed consumption rates: record adds doses and residue doses every day.
4.3 healthy state: observe piglet healthy state every day, record piglet death condition.
5. experimental result
As seen from the above table, each growth indexes of experimental group sucking piglets all makes moderate progress than control group.
1) sucking piglets wean bacterial classification (individuality): control group counterpoise is 7.75kg, experimental group 8.25kg.Experimental result: experimental group individuality wean counterpoise comparatively control group improves 6.45%.
2) average daily gain (individuality): control group day weight gain 178.7g, experimental group day weight gain 187.5g.The i.e. experimental group comparatively individual average daily gain raising 4.92% of control group.
3) average individual day feed consumption: control group day feed consumption 42.4g, experimental group day feed consumption 39.2g.The i.e. individual average day feed consumption of experimental group comparatively control group reduction by 7.55%.
4) feed-weight ratio: control group feed-weight ratio is 0.221, experimental group feed-weight ratio is 0.209, i.e. experimental group feed-weight ratio comparatively control group reduction by 5.43%.
5) mortality ratio: control group is 19.68%, experimental group is 12.07%.I.e. experimental group mortality ratio comparatively control group reduction by 38.67%.
In sum, the present invention is separated and obtains from healthy raw material, through being accredited as lactobacillus reuteri LZ262.Described lactobacillus reuteri LZ262 has good tolerance to human body and animal body gastrointestinal environment, and proteolytic enzyme can be produced help digest, good symplastic growth effect is had with milk-acid bacteria, expand the range of application of this bacterium, fermentation has higher viable bacteria amount, decrease fermentation costs, and sucking piglets growth performance can be significantly improved.Described lactobacillus reuteri LZ262 is used for the fields such as animal feedstuff additive, antibacterial liquid, food, healthcare products and cultivation.
The above, it is only preferred embodiment of the present invention, not any pro forma restriction is done to the present invention, although the present invention discloses as above with preferred embodiment, but and be not used to limit the present invention, any those skilled in the art, do not departing within the scope of technical solution of the present invention, make a little change when the technology contents of above-mentioned announcement can be utilized or be modified to the Equivalent embodiments of equivalent variations, in every case be the content not departing from technical solution of the present invention, according to technical spirit of the present invention to any simple modification made for any of the above embodiments, equivalent variations and modification, all still belong in the scope of technical solution of the present invention.
Claims (10)
1. a lactobacillus reuteri, also lactobacillus reuteri is claimed, it is characterized in that: it is lactobacillus reuteri (Lactobacillusreuteri) LZ262, preserving number is CGMCCNo.11240, preservation date: on 08 14th, 2015, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center.
2. lactobacillus reuteri according to claim 1, is characterized in that: have higher viable bacteria amount in described lactobacillus reuteri LZ262 substratum after optimization.
3. lactobacillus reuteri according to claim 1, is characterized in that: described lactobacillus reuteri LZ262 has tolerance to people and animal hydrochloric acid in gastric juice and cholate.
4. lactobacillus reuteri according to claim 1, is characterized in that: described lactobacillus reuteri LZ262 has higher survival after spraying dry.
5. lactobacillus reuteri according to claim 1, is characterized in that: described lactobacillus reuteri LZ262 has stronger biocidal property to intestinal bacteria.
6. lactobacillus reuteri according to claim 1, is characterized in that: described one of lactobacillus reuteri LZ262 and following milk-acid bacteria or more than one are composite, has symplastic growth effect;
Lactobacterium acidophilum (Lactobacillusacidophius);
Lactobacillus delbrueckii kind (Lactobacillusdelbrueckiisubsp.bulgaricus);
Casei subspecies (Lactobacilluscaseisubsp.Casei);
Thermophilus streptococcus (Streptococcusthermophius);
Streptococcus acidi lactici (Streptococcuslactis);
Lactococcus lactis subsp.lactis (Lactococcuslactissubsp.lactis);
Plant lactobacillus (Lactobacillusplantarum);
Bifidobacterium animalis subspecies (Bifidobacteriumanimalissubsp.lactis).
7. lactobacillus reuteri according to claim 1, is characterized in that: described lactobacillus reuteri LZ262 has stronger enzymatic productivity.
8. an application for lactobacillus reuteri as claimed in claim 1, is characterized in that: described lactobacillus reuteri LZ262 is used for animal feedstuff additive, antibacterial liquid, food, healthcare products.
9. the application of lactobacillus reuteri according to claim 8, it is characterized in that: be that the ratio between wherein said lactobacillus reuteri LZ262 and other probiotic bacterium is: lactobacillus reuteri LZ262: Lactobacillus acidophilus: lactobacterium casei: plant lactobacillus=(1 ~ 2): (3 ~ 5): (2 ~ 3): (1 ~ 2) by described lactobacillus reuteri LZ262 and other probiotic bacterium assembly when described lactobacillus reuteri LZ262 is used for animal feedstuff additive.
10. the application of lactobacillus reuteri according to claim 8 or claim 9, is characterized in that: described lactobacillus reuteri LZ262 is used for animal feedstuff additive to carry out growing and fattening pigs and feed.
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